A New, Rapid Method for the Quantification of Dolichyl Phosphates in Cell Cultures Using TMSD Methylation Combined with LC–MS Analysis

Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes. They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors. These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatography–high-resolution mass spectrometry (LC–HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance. To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis. The analytical method was validated for its reproducibility, sensitivity, and accuracy. The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and Saccharomyces cerevisiae cells.

Dolichyl phosphates (DolP) are ubiquitous lipids that are present in almost all eukaryotic membranes.They play a key role in several protein glycosylation pathways and the formation of glycosylphosphatidylinositol anchors.These lipids constitute only ~0.1% of total phospholipids, and their analysis by reverse phase (RP) liquid chromatographyhigh-resolution mass spectrometry (LC-HRMS) is challenging due to their high lipophilicity (log P > 20), poor ionization efficiency, and relatively low abundance.To overcome these challenges, we have introduced a new approach for DolP analysis by combining trimethylsilyldiazomethane (TMSD)-based phosphate methylation and HRMS analysis.The analytical method was validated for its reproducibility, sensitivity, and accuracy.The established workflow was successfully applied for the simultaneous characterization and quantification of DolP species with different isoprene units in lipid extracts of HeLa and Saccharomyces cerevisiae cells.

Background
Dolichyl phosphates (DolP) are long-chain polyisoprenoid phosphates that function as glycan carriers in cellular Nand O-glycosylation, C-and O-mannosylation, and glycosylphosphatidylinositol-anchor biosynthesis (Orlean, 1990;Rosenwald et al., 1990).Numerous studies have reported altered endogenous DolP levels in pathophysiological conditions such as Alzheimer's disease, dementia, and Prion disease (Kranz et al., 2007;Park et al., 2014;Sabry et al., 2016;Jaeken et al., 2020).Given the essential role of DolP in cellular glycosylation, there is a need for a rapid method to accurately characterize and measure DolP in biological samples, including those from patients with disorders associated with glycosylation defects.Generally, DolP are quantified after their fluorescence derivatization by combining normal-phase liquid chromatography (LC) with fluorescence detection (Haeuptle et al., 2010).This method, based on a multi-step sample preparation and derivatization procedure, is labor intensive and time consuming.Few mass spectrometry (MS)based methods exist for the structural characterization of endogenous DolP species (Löw et al., 1991;Wolucka et al., 1996;Guan et al., 2010;Taguchi et al., 2016).To date, a method for the simultaneous profiling and quantification of DolP species of different isoprene chain lengths in biological membranes has been lacking.We have developed a method for the characterization and quantification of DolPs using trimethylsilyldiazomethane (TMSD) derivatization followed by reverse phase (RP) liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis.TMSD is frequently used as a methylating agent for anionic phospholipids (Lee et al., 2017) and free fatty acids (Mok et al., 2016) to increase the electrospray ionization efficiency.The scheme of the methylation reaction of DolP is illustrated in Figure 1.We found that TMSD methylation enabled chromatographic retention of DolPs in RPLC analyses.In the Tandem MS (MS/MS or MS2) analysis of all methylated DolP species by high-energy collisional dissociation, the most intense fragment ion at m/z 127.0155 represents a characteristic headgroup fragment, corresponding to a dimethylphosphate group.Transitions from all parent m/z (Table 1) to 127.0155 in the MS2 spectra were used to determine the identification of methylated DolPs and dodecaprenyl phosphate (PolP C60).The established workflow uses a fast and efficient single-step derivatization procedure.Furthermore, the method Recipes 1. 15 M KOH Accurately weigh 8.4 g of KOH pellets into a 100 mL volumetric flask.Add LC-grade water dropwise with intermittent slow vortex to dissolve KOH.Be careful, as an exothermic reaction will occur, and the reaction flask will be hot.After complete dissolution, dilute the solution to the mark.Use a glass bottle with a PTFE lid for storage of the KOH solution.

10 M AmAc
Take the 10 mL volumetric flask along the glass stopper and rinse both twice with MS-grade methanol.Dry them carefully under Argon.Transfer accurately weighed 7.708 g of LC-MS grade AmAc to the volumetric flask.To dissolve the solid, add distilled water in steps of approximately 100 µL, followed by sonication.Once all solids are dissolved, fill the volumetric flask to the etched line.This stock can be stored at 4 °C for up to six months.
3. Solvent A: acetonitrile/water 60:40 (v/v) with 0.1% FA and 10 mM AmAc Prepare 1,000 mL of solvent mixture of acetonitrile/water 60:40 (v/v) and add 1 mL of formic acid and 1 mL of 10 mM AmAc stock.Mix solvent mixture thoroughly and degas for 5 min in an ultrasonic water bath sonicator.
4. Solvent B: isopropanol/water 90:10 (v/v) with 0.1% FA and 10 mM AmAc Prepare 1,000 mL of solvent mixture of isopropanol/water 90:10 (v/v) and add 1 mL of FA and 1 mL of 10 mM AmAc stock.Mix solvent mixture thoroughly and degas for 5 min in an ultrasonic water bath sonicator.

Procedure
A schematic of the workflow for the analysis of DolPs is shown in Figure 2. The different procedures involved in this workflow are mentioned below.4. Add 1 mL of methanol and vortex for 1 min at normal speed, followed by sonication for 1 min in an ultrasonic water bath sonicator.5. Add 1 mL of water and vortex for 1 min at normal speed, followed by sonication for 1 min with an ultrasonic water bath sonicator.6. Centrifuge at 3,500× g for 5 min at RT. Note: If you wish to normalize DolP amounts to total lipid/total phosphatidylcholine (PC) (as bulk membrane lipid), measure the total lipid/PC levels of samples at this point.See Özbalci et al. (2013) for the procedure to determine the total lipid/PC levels of samples (Özbalci et al., 2013).

Procedure for alkaline hydrolysis and extraction
Note: Use round bottom screw-top glass tubes with Teflon-lined caps for DolP sample preparation.Dichloromethane (which is added in subsequent steps) can dissolve plastic materials.Therefore, always use glass bottles, pipettes, and syringes while working with dichloromethane.1. Fill the water bath with distilled water to the upper level of 2.5 mL of solvent in a glass tube immersed in the water bath.Heat the water bath to 85 °C.2. Add 0.5 mL of 15 M KOH to samples to initialize alkaline hydrolysis.3. Vortex for 1 min, followed by centrifugation at 3,500× g for 5 min at RT. 4.Then, immerse glass tubes in a preheated linear shaking water bath at 85 °C for 1 h, under constant shaking at 40 rpm.Note: This strong alkaline treatment releases DolP from DolP-linked monosaccharides, dolichyl diphosphate (DolPP)-linked mono-and oligosaccharides, and DolPP (Keller et al., 1985;Lee Adair and Kennedy Keller, 1985;Fernandez et al., 2001;Schenk et al., 2001). 5. Cool samples to RT. Vortex for 1 min, followed by centrifugation at ~3,500× g for 5 min at RT. 6. Increase the level of water in the water bath with distilled water to the upper level of 7.5 mL of solvent in a glass tube immersed in the water bath.Heat the water bath to 40 °C.
7. To the cooled samples, carefully add 1 mL of methanol and 4 mL of dichloromethane to induce phase partitioning.8.Then, immerse glass tubes in a preheated linear shaking water bath at 40 °C for 1 h, under constant shaking at 40 rpm.9. Cool the samples at RT; then, vortex and centrifuge at 3,500× g for 5 min at RT. 10.Following centrifugation, remove the upper aqueous phase using a Pasteur pipette and wash the lower organic phase four times with wash solvent (see Recipes).11.Carefully transfer the bottom layer to a new glass tube using a Pasteur pipette.12. Dry samples under a gentle stream of nitrogen at RT in a Turbovap evaporator.

Methylation of DolPs
Note: Inhaling TMSD may cause lung injury or central nervous system depression.TMSD must be handled with caution and following proper safety standards.Before starting the reaction, ensure that the hood is working properly.When working with TMSD, personal safety equipment such as lab aprons, latex gloves, goggles, and respirators should be worn at all times.Screw cap glass tubes immediately after the addition of the reagents.1. QC standard: methylate DolP standard and IS mixture along with samples.In a screw-top glass tube, take 20 pmol of DolP standard and 20 pmol of IS and dry under a gentle stream of nitrogen.2. Add 200 μL of dichloromethane:methanol mixture (6.5:5.2, v:v, see Recipes) to dried samples.3. Vortex for 1 min followed by centrifugation at 3,500× g for 5 min at RT. 4. To these tubes, carefully add 10 μL of TMSD using a gas-tight Hamilton syringe. 5. Vortex for 1 min, followed by centrifugation at 3,500× g for 5 min at RT. Note: The color of the solution should change from yellow to colorless after the addition of acetic acid.Add acetic acid in 5 μL increments until the yellow color disappears.8. Dry samples under a gentle stream of nitrogen at RT in a Turbovap evaporator.9.For LC-HRMS analysis, reconstitute dried samples in 120 μL of methanol.Vortex for 1 min and transfer lipid films to micro inserts placed in 1.5 mL Eppendorf tubes.10.Centrifuge the samples in Eppendorf tubes in a microcentrifuge at 1,500× g for 5 min at 4 °C .Transfer micro inserts to LC vials for analysis.

LC system preparation
1. Prepare Solvents A and B according to Recipes. 2. Mix each solvent thoroughly by shaking and degas both solvents in the water bath sonicator with loose caps for at least 10 min.3. Put up both solvents on the LC system and purge all lines for 5 min.4. Set the flow rate to 0.1 mL/min with both solvents in the ratio 60:40 (solvent A:B). 5. Maintain the column at 55 °C.6. Equilibrate the column for at least 15 min after the column pressure is stable.7. Set the LC gradient as follows: 40% B at 0 min, 40%-50% B (0-3 min), 50%-54% B (3-9 min), 54%-70% B (9-9.1 min), 70%-90% B (   5. All methylated DolP species and IS PolP C60 exhibited a characteristic fragment of 127.0155, which is used as a qualifier ion (assigned to the dimethylphosphate group), independent of dolichol or isoprenyl chain length.6. Endogenous DolP species were identified using matching MS1, retention time, and MS2 spectrum to standards.

Quantitative analysis of DolP in samples
1. MS data quantification was established in Quanbrowser by creating a processing method using XCalibur Quanbrowser (Thermo Fisher Scientific XCalibur 3.1 Quanbrowser User Guide, 2014) 2. Get the peak area from the XICs of DolPs and its internal standards based on its accurate m/z value with an accuracy of 10 ppm.The measured peak areas of the methylated DolPs and internal standards must be corrected by multiplying the isotope correction factors.This correction will take into account the differences in the relative 13C natural isotope abundance of different DolP species.3. Theoretical isotopic distributions were obtained using the online EnviPat R package Tool (Loos et al., 2015).
Finally, the concentration of DolPs was estimated by comparing the peak areas of internal standards with known concentrations to the peak areas of DolPs.

6 .
Incubate for 40 min at RT. Note: The chemical reaction of TMSD (yellow) with the acetic acid produces (colorless) methyl acetate.After adding TMSD to the lipid extracts, the solution should have a faint yellow color.If the yellow color associated with TMSD disappears in the solution, add TMSD in 5 μL increments until a yellow color appears.It is recommended to dry the lipid extracts completely before methylation to avoid neutralization of TMSD.7. When the reaction is complete, neutralize excess TMSD by slowly adding 10 μL of acetic acid.

Figure 4 .
Figure 4. Identification of dolichyl phosphates (DolP) C95 in DolP standard mixture (left panel) and in HeLa cell extracts (right panel) based on A) extracted ion chromatograms (XIC), B) MS spectrum, and C) MS2 spectrum of [M+NH4] + ions of DolP C95.For more MS spectral characterization and protocols, please refer to Kale et al. (2023).